Comparative analysis of expression of histone H2a genes in mouse

Background At least 18 replication-dependent histone H2a genes are distributed in 3 Hist gene clusters on different chromosomes of the mouse genome. In this analysis we designed specific PCR primers for each histone H2a transcript and studied the expression levels and patterns using quantitative RT-PCR (qRT-PCR). In addition, we compared histone H3 K9 acetylation levels in the promoter regions of H2a genes by ChIP (chromatin immunoprecipitation) – quantitative PCR (qPCR) analysis. Results RT-PCR analysis indicated that all 20 histone H2a genes assessed in this study are expressed. The replication-dependent histone H2a genes have different expression levels but similar expression patterns. Among the 20 histone H2a genes, the expression-level of H2afz, a replication-independent gene, was highest, and that of Hist1h2aa, a replication-dependent gene, was lowest. Among 18 replication-dependent H2a genes, the expression level of Hist3h2a was highest. The ChIP-qPCR analysis showed that histone H3 K9 acetylation levels in promoter regions of both H2afz and Hist3h2a are clearly higher than that in the promoter region of Hist1h2aa. The H3 K9 acetylation level in the promoter of Hist1h2aa is similar to that in the γ-satellite region. Conclusion These results strongly suggest that histone H3 K9 acetylation plays a role in the expression of histone genes.


Background
Eukaryotic genomic DNA is packaged with chromosomal proteins, forming chromatin. The most fundamental repeating unit of chromatin is the nucleosome. The nucleosome core consists of 146 bp of DNA wrapped around an octamer of histone proteins made up of 2 copies each of histones H2A, H2B, H3, and H4, in 1.65 turns [1]. Replication of the eukaryotic chromosomes requires the synthesis of histones to package the newly replicated DNA into chromatin. Control of the level of histone mRNA accounts for much of the control of histone protein synthesis [2]. It is still an open question as to how the expression of individual histone genes is controlled.
The variants and modifications of the histone proteins are related to chromatin structure [3][4][5][6]. Specific amino acids within histone tails are targets for a number of post-transcriptional modifications, i.e., acetylation, methylation, phosphorylation, and ubiquitination [3]. In particular, the modification of histone H3 K9 affects chromatin structure. H3 K9 methylation is enriched in transcriptionally silent genes and heterochromatin. On the other hand, H3 K9 acetylation is enriched in transcriptionally active genes [7]. Is this modification related to histone gene expression?
Eighteen replication-dependent histone H2a genes were identified in the mouse genome sequence [8]. Among these 18 genes, 13 are located in the Hist1 cluster on chromosome 13, 4 in the Hist2 cluster on chromosome 3, and 1 in the Hist3 cluster on chromosome 11 [8]. Thus, replication-dependent histone H2a genes are distributed in at least 3 Hist clusters. In addition, the mouse has 2 replication-independent histone H2a genes, H2afx on chromosome 9 and H2afz on chromosome 3. Recently we reported a novel replication-independent histone H2a gene (H2afj) on chromosome 6 [9]. H2afz and H2afj are typical replication-independent genes [9,10]. The H2afz protein is enriched in euchromatic regions and acts synergistically with a boundary element to prevent the spread of heterochromatin [6]. On the other hand, H2afx mRNA has both a polyadenylated tail and a stem-loop structure [11], elements typical of, respectively, replication-independent and replication-dependent histone genes.
As cells progress from G1 to S phase, the rate of histone gene transcription increases 3-to 5-fold, and the efficiency of histone pre-mRNA processing increases 8-to 10-fold, resulting in a 35-fold increase in histone protein levels [2,12]. Most promoters of histone genes have CCAAT and TATA boxes [9,13]. Some promoters have an E2F binding motif between the CCAAT and TATA boxes. This E2F binding motif is recognized, and then the E2F transcription factor activates an H2a gene in early S-phase of the cell cycle [14]. However, it is not known how transcription-related proteins cooperate to coordinately regulate histone gene transcription during the cell cycle.
The amino acid sequences of histone H2a proteins are very similar, except for that of H2afz protein [9]. For example, Hist1h2ab, 2ac, 2ad, 2ae, 2ag, 2ai, 2an, and 2ao encode the same structural protein. Among these 8 genes, Hist1h2ad and 2ao have the same nucleotide sequence; however, the others have different nucleotide sequences. Quantitative RT-PCR analysis can be used to show the expression levels of different genes (for example [15]). Thus, in this study we designed the specific PCR primers for each histone H2a gene and studied the expression levels and patterns by qRT-PCR.

Results and discussion
Each product of the qRT-PCR gave a single band on the agarose gel, located in the expected position ( Fig. 1). This result indicates that all histone H2a genes are expressed in Hepa 1-6 cells. The expression levels of 18 replicationdependent histone genes and H2afx increased along with cell cycle progression from the beginning (0 h) of S-phase to the middle (2-4 h) of S-phase, and then decreased from the middle to the end (6 h) of S-phase (Fig. 2). On the other hand, the expression level of the replicationindependent gene H2afz lacked such a single peak during H2afz is regulated in a replication-independent manner, but H2afx is regulated in a replication-dependent manner. This pattern is consistent with the results of a previous report that indicated that H2afx gives rise to a cell-cycleregulated mRNA ending in the stem-loop during S-phase, and a polyadenylated mRNA during G1-phase [10]. Therefore, H2afx is regulated in a replication-dependent manner (Fig. 2). On the other hand, H2afz lacks regulation of a polyadenylated mRNA. Interestingly, expression levels of H2afz decreased at the end (6 h) of S-phase, similar to those of replication-dependent genes (Fig. 2). This result suggests that the decrease at the end of S-phase is independent of the histone H2a mRNA structure.
Among the 13 genes in the Hist1 cluster, the expression level of Hist1h2ae was approximately 100 times that of Hist1h2aa (Fig. 3). In addition, the 4 genes in the Hist2 cluster had different expression levels. Thus, the expression level of Hist2h2aa1/2aa2 was approximately 10 times that of Hist2h2ab/2ac (Fig. 3). Therefore, the expression levels of genes belonging to the same gene cluster were different.
One possibility is that such different expression levels are caused by different promoters and different binding proteins bound to the promoters. For example, the promoters of Hist1h2ad, Hist1h2af, Hist1h2ag, and Hist1h2ah have the E2F binding motif (5'-TTTTCGCGCCC-3') between the CCAAT and TATA boxes [9]. Among these 4   replication-dependent genes, the expression level of Hist1h2ah was approximately 10 to 20 times that of Hist1h2ag (Fig. 3). In addition, compared among all 20 genes assessed in this paper, the expression levels of H2afz, Hist3h2a, Hist2h2aa1/2aa2, Hist1h2ae, and Hist1h2ai/aj were higher than that of Hist1h2ah, and those of Hist1h2ak and Hist1h2aa were lower than that of Hist1h2ag (Fig. 3). Thus, the relation between the E2F binding motif and the expression level is not clear. Unfortunately, we cannot determine here which structure of the promoters causes such different expression levels.

Expression patterns and levels: results of a) first and b) second qRT-PCR analyses
Next, we compared the histone H3 K9 acetylation levels in the promoter regions of H2afz (highest expression), Hist3h2a (highest expression among replication-dependent H2a genes), and Hist1h2aa (lowest expression). The ChIP-qPCR analysis showed that histone H3 K9 acetylation levels in the promoter regions of both H2afz and Hist3h2a were clearly higher than that in the promoter region of Hist1h2aa. The H3 K9 acetylation level in the promoter of Hist1h2aa was similar to that in the γ-satellite heterochromatin region (Table 1). This result indicates that the expression of histone H2a genes is related to the acetylation of histone H3 K9 in the promoter region.

Conclusion
This study strongly suggests that histone H3 K9 acetylation plays a role in the expression of histone genes.

Cell cycle synchronization
The cell cycle of mouse Hepa 1-6 cells was synchronized at the end of G1-phase by the addition of thymidinehydroxyurea. The cell cycle arrest was released by washing out the thymidine-hydroxyurea, then the cells were harvested at intervals of 1 h from 0 to 13 h.

RNA extraction
Total RNA was extracted by using the RNeasy mini kit (Qiagen) according to the instructions in the manual for

Quantitative PCR
The primers used in this analysis are shown in Table 2. Quantification of GAPDH (glyceraldehyde-3-phosphate dehydrogenase) mRNA (primers 5'-TGTGTCCGTCGT-GGATCTGA-3' and 5'-CCTGCTTCACCACCTTCTTGA-3' ; product size 76 bp) was used as a control for data normalization. PCR amplification was performed on an ABI PRISM 7700 Sequence Detection System (Applied Biosystems). The PCR conditions were an initial step of 30 s at 95°C, followed by 40 cycles of 5 s at 95°C and 30 s at 60°C. The SYBR premix Ex Taq (Takara) was used according to the manual. Each amplification curve was checked [16]. Expression was assessed by evaluating threshold cycle (C T ) values. The relative amount of expressed RNA was calculated by using Livak and Schmittgen's method [17]. The qRT-PCR analyses were performed twice. In each analysis, we adjusted the H2afz expression level to 1 at 0 h.